Boosting DNA vaccine power by lipid nanoparticles surface engineered with amphiphilic bioresorbable copolymer

Successful DNA vaccination generally requires the aid of either a viral vector within vaccine components or an electroporation device into the muscle or skin of the host. However, these systems come with certain obstacles, including limited transgene capacity, broad preexisting immunity in humans, and substantial cell death caused by high voltage pulses, respectively. In this study, we repurposed the use of an amphiphilic bioresorbable copolymer (ABC), called PLA-PEG, as a surface engineering agent that conciliates lipid nanoparticles (LNPs) between stability during preparation and biocompatibility post-vaccination. The LNP carrier can be loaded with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific DNA; in this form, the DNA-LNP is immunogenic in hamsters and elicits protective immunity following DNA-LNP vaccination against heterologous virus challenge or as a hybrid-type vaccine booster against SARS-CoV-2 variants. The data provide comprehensive information on the relationships between LNP composition, manufacturing process, and vaccine efficacy. The outcomes of this study offer new insights into designing next-generation LNP formulations and pave the way for boosting vaccine power to combat existing and possible emerging infectious diseases/pathogens.


Supplemental methods
In vitro imaging.After 72-hr transfection with GFP DNA-LNPs, the transfected cells were cultured in 24-well plates (Cat. #142475, Nunc, Thermo-Fisher Scientific, China) in 1 mL culture medium, and images were taken with a fluorescence microscope (Olympus IX73 inverted microscope, Tokyo, Japan).Cell nuclei were stained with Hoechst 33342 dye (Thermo-Fisher Scientific, Rockford, IL, USA) before imaging.
In vitro luminescence.After 72-hr transfection with CBGr99 DNA-LNPs, the transfected cells were harvested and washed with PBS and then lysed using 150 µL of Passive Lysis Buffer (Cat.#E1941, Promega, WI, USA).The luciferase activity was measured by mixing 50 µL of the cell lysate with 50 µL of luciferase substrate (Cat.#E151A, Promega, WI, USA) in a 96-well white plate (Cat.#236108, Nunc, Thermo-Fisher Scientific, Denmark) and then measuring the luminescence by an Orion L Microplate Luminometer (Berthold Detection System, TN, USA).
Luciferase activities were expressed as relative light units per second (RLU/s).
RNA extraction and RT-qPCR.RNA was extracted at 24, 48, 72 hrs post-transfection using the RNeasy ® Mini Kit (Cat.#74104, QIAGEN, Hilden, Germany) following the manufacturer's protocol.Prior to RNA extraction, the cell culture medium was removed, and the cells were washed once with PBS before being lysed.From the lysates (600 ng of RNA), cDNA was synthesized using SuperScript III Reverse Transcriptase (Cat.#18080044, Thermo-Fisher Scientific, MA, USA) with oligo(dT)12-18 primer (Cat.#18418012, Thermo-Fisher Scientific, MA, USA) following the manufacturer's instructions.Before qPCR reaction, cDNA samples were diluted 3 times in RNase-and DNase-free water. 1 µL cDNA was used in each reaction with 200 nM forwards and reverse primers (Integrated DNA Technologies, Singapore).PCR was performed with KAPA SYBR ® FAST qPCR Master Mix (Cat.#kk4609, KAPA BIOSYSTEMS, MA, USA) on a LightCycler ® 480 (Roche Diagnostics, IN, USA) at a total volume of 10 µL in 384-well plates (Roche Diagnostics, IN, USA).The cycling conditions were set as pre-incubation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for 1 sec.The program ended with melting curve analysis to check primer dimers.In the experiment, two replicates of cDNA samples were performed along with no transcriptase control or no template control to check genomic DNA contamination.Expression data were analyzed using the Delta-Delta Ct method.
The expression of GAPDH was used as a reference control.
DNA-LNP stability.Stability monitoring of the DNA-LNP formulations was performed at 4, 15, 25, and 37°C.At week 2 and week 4, three specimens were withdrawn and tested for their particle size distribution, encapsulation efficiency and transfection of DNA reporter genes in HEK293 cells (human embryonic kidney 293 cells, Cat.#BCRC60019).
In vivo luminescence in mice.BALB/c mice were obtained from the NLABRC (Taipei, Taiwan) and housed at the Animal Center of the NHRI and maintained in accordance with institutional animal care protocols (Protocol No: NHRI-IACUC-109077-A). Transfection of DNA molecules and expression of luciferase protein in mice were measured by using in vivo luminescence.Mice were inoculated with predetermined amounts of the DNA-LNP (10 µg per mouse) via the intramuscular (i.m.) route.On Days 1, 2, 3, 4, 7, 10, 14, 21, and 28 after administration, mice were injected intraperitoneally (i.p.) with 100 µL D-luciferin (Cat.# ab143655, Abcam, Cambridge, UK) at a concentration of 15 mg/mL in PBS, and the reaction were performed for 11 minutes.
Luminescence signals were collected by an IVIS Spectrum instrument (Perkin Elmer, Waltham, MA, USA), and the luminescence signals in regions of interest (ROIs) were quantified using the imaging software "Living Image".

Figure S2 .
Figure S2.Effects of LNP on DNA transfection in the cell culture system 1 × 10 5 HEK293 cells were treated with GFP-encoding plasmid DNA, either in culture medium

Figure S3 .
Figure S3.Transfection of DNA-LNP formulation in vitro and in vivo

Figure S4 .
Figure S4.Impact of the lipid contents on the transfection of DNA-LNP formulation in the

Figure S6 .
Figure S6.Omicron TS-specific IgG titers in hamsters injected with a two-dose TSomi DNA-